U.S. patent number 5,242,915 [Application Number 07/885,864] was granted by the patent office on 1993-09-07 for formulation useful for the inhibition of massive release of cerebral glutamate.
This patent grant is currently assigned to Shionogi & Co., Ltd.. Invention is credited to Ikuo Adachi, Masami Eigyo, Takefumi Gemba, Motohiko Ueda.
United States Patent |
5,242,915 |
Ueda , et al. |
September 7, 1993 |
Formulation useful for the inhibition of massive release of
cerebral glutamate
Abstract
A method useful for the treatment or prevention of diseases
caused by a massive release of cerebral glutamate, in particular
epilepsy and dementia, and pharmaceutical formulations therefor,
which comprise, as an active ingredient, a therapeutically
effective amount of methyl
(+)-(4S)-4,7-dihydro-3-isobutyl-6-methyl-4-(3-nitrophenyl)thieno[2,3-b]pyr
idine-5-carboxylate (S-312-d).
Inventors: |
Ueda; Motohiko (Suita,
JP), Gemba; Takefumi (Amagasaki, JP),
Eigyo; Masami (Ikoma, JP), Adachi; Ikuo (Suita,
JP) |
Assignee: |
Shionogi & Co., Ltd.
(Osaka, JP)
|
Family
ID: |
15416271 |
Appl.
No.: |
07/885,864 |
Filed: |
May 20, 1992 |
Foreign Application Priority Data
|
|
|
|
|
May 21, 1991 [JP] |
|
|
3-146820 |
|
Current U.S.
Class: |
514/301 |
Current CPC
Class: |
A61K
31/44 (20130101); A61P 25/08 (20180101); A61P
9/10 (20180101); A61P 25/28 (20180101); A61P
25/00 (20180101) |
Current International
Class: |
A61K
31/44 (20060101); A61K 031/395 () |
Field of
Search: |
;514/210 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Chem. Abst. 114-35719.RTM.(1991). .
Scriabine et al., The FASEB Journal, vol. 3, May 1989 pp.
1799-1806. .
Tollefson, Biol. Psychiatry, vol. 27, No. 10, May 15, 1990 pp.
1133-1142. .
Masui et al., Drug Development Research, vol. 20, 1990 pp.
453-464..
|
Primary Examiner: Friedman; S. J.
Attorney, Agent or Firm: Wenderoth, Lind & Ponack
Claims
What is claimed is:
1. A method for treating a disease accompanied by or caused by a
massive release of cerebral glutamate, which comprises
administering a therapeutically effective amount of methyl
(+)-(4S)-4,7-dihydro-3-isobutyl-6-methyl-4-(3-nitrophenyl)thieno[2,3-b]pyr
idine-5-carboxylate to a patient in need of such treatment.
2. The method as claimed in claim 1, wherein the disease is
epilepsy.
3. The method as claimed in claim 1, wherein the disease is
dementia.
4. The method as claimed in claim 1, wherein the disease is of
ischemic neuronal cell damages.
Description
FIELD OF THE ART
The present invention relates to pharmaceutical formulations
capable of inhibiting a massive release of cerebral glutamate,
which comprise, as an active ingredient, methyl
(4S)-4,7-dihydro-3-isobutyl-6-methyl-4-(3-nitrophenyl)thieno[2,3-b]pyridin
e-5-carboxylate, and a method for preventing and treating diseases
caused by the massive release of cerebral glutamate. More
specifically, it relates to pharmaceutical formulations useful for
the treatment or prevention of epilepsy, dementia, or necrosis of
cerebral cells.
BACKGROUND OF THE INVENTION
Recently, interest has been concentrated on various types of
dementia such as cerebrovascular dementia, Altzheimer's disease,
senile dementia, and the like, and the demand for effective methods
for treating and preventing them are continuously increasing. It
has been reported that a neurotoxic effect of cerebral excitatory
amino acids, especially glutamate, which exhibits a neurotoxic
effect following the binding to receptors, may be responsible for
these diseases (Sinozaki, Gendaikagaku, 10: 38-44 (1987)). Prior to
the present invention, many compounds had been provided with the
intention of decreasing or eliminating the above aggravating effect
of cerebral glutamate, including N-methyl-D-aspartic acid
(hereinafter referred to as NMDA) channel antagonist such as MK-801
(H. Kato et al., Brain Res. 516: 175-179 (1990)]. However, they are
not clinically applicable because of serious toxic effects.
Therefore, safe compounds capable of preventing the massive release
of cerebral glutamate have been required.
In the course of an investigation into dihydropyridine derivatives
for the purpose of developing substances having Ca-blocking
activity, a series of
4-aryl-4,7-dihydrothieno[2,3-b]pyridine-5-carboxylate derivatives
were synthesized and tested for the binding affinity to Ca.sup.++
channels, coronary vasodilating effect and antihypertensive
activity, and methyl
4,7-dihydro-3-isobutyl-6-methyl-4-(3-nitrophenyl)thieno[2,3-b]pyridine-5-c
arboxylate (hereinafter, referred to as S-312) was found to be a
potent vasodilating agent and also proved to have a potent
antihypertensive and coronary vasodilating effect with lesser
adverse reactions (U.S. Pat. No.4,703,051). Further study revealed
that the major biological activities of said compound resides in
the (+)-enantiomer with S configuration, i.e., methyl
(+)-(4S)-4,7-dihydro-3-isobutyl-6-methyl-4-(3-nitrophenyl)thieno[2,3-b]pyr
idine-5-carboxylate (hereinafter, referred to as S-312-d), whose
preparation is described in the Japanese Patent Publication (KOKAI)
No.52890/1992.
Because of their selective Ca-blocking effect on vascular smooth
muscle, S-312-d and S-312 exhibit antihypertensive activity, as
well as peripheral, cerebral, and coronary vasodilating actions,
which makes them useful in the treatment of circulatory diseases
such as angina pectoris, hypertension, cerebrovascular dysfunction,
arrhythmia, or the like. The useful effect on various
cerebrovascular diseases is however not attributable to any
specific properties of these compounds, but to the cerebral
vasodilating effect common to Ca-blockers. Though one of ordinarily
skill in the art might have thought that Ca-blockers were effective
for preventing the cerebral apoplexy from occurring based upon
their vasodilating action, he could hardly expect before the
present invention that S-312-d was so effective on the ischemic
neuronal cell damages consequent upon the cerebral apoplexy as well
as the senile dementia.
DISCLOSURE OF THE INVENTION
The present invention provides a pharmaceutical formulation useful
in the prevention or treatment of various diseases caused by a
massive release of cerebral glutamate. Examples of such diseases
include epilepsy, senile dementia, Huntington's chorea, neuronal
cell damage following ischemia, and the like. The details of the
invention will be hereinafter described.
The present invention was established by the finding that S-312-d
has an ability of inhibiting specifically a massive release of
cerebral glutamate from hippocampus, which phenomenon is often
observed during cerebral ischemia. Further, S-312-d was proved to
have a potent inhibitory effect on a convulsion induced by sound
stimulation in DBA/2 mice but not on those induced by NMDA or
pentylenetetrazol.
The effect of S-312-d on the massive release of cerebral glutamate
was investigated using stroke-prone spontaneously Hypertensive Rats
(hereinafter, referred to as SHRSP). Thus, an experimental cerebral
ischemia was caused in SHRSPs by the occlusion of both common
carotid arteries (hereinafter, referred to as BCAO). During the
ischemic period, a massive release of glutamate in hippocampal CAI
region was observed (see, FIG. 1). S-312-d and the other
dihydropydine Ca.sup.++ antagonists (nimodipine, nilvadipine) were
then evaluated for inhibitory effect on the ischemic glutamate
release in hippocampal CAI region, resulting in that only S-312-d
was significantly effective (see, FIG. 2). It is noteworthy that
S-312-d inhibits exclusively the ischemic glutamate release but not
the normal resting release.
According to the recent reports, cerebrovascular dementia,
Altzheimer's disease, Huntington's disease, and the like are
probably associated to neurotoxic effects of cerebral excitatory
amino acids, typically glutamate. There are some proposals as to
the mechanisms by which glutamate exerts its neurotoxic effect.
Cotman, C. W. and Iversen, L. L. (TINS, 10: 263-265 (1987))
suggested that the amino acid-release causes an over excitement of
NMDA receptor, a subtype of glutamate receptor, which leads to a
progressive pathological change and the death of neuronal
cells.
According to the proposal of Nagasawa and Kogure ("IGAKU-NO-AYUMI",
vol. 158, pp.613-618, Aug. 31, 1991)), the cause of a delayed
necrosis of neuronal cells can be explained as follows: when
cerebral cells are faced with an ischemic condition, (a massive
calcium influx takes place following the ischemic depolarization of
neuronal cell membrane), and which leads to the delayed cellular
necrosis.
As can be seen from the experimental results shown in Table 2,
S-312-d possesses an ability of preventing the delayed necrosis of
neuronal cells following ischemia and is useful for protecting
cranial nerve neuronal cells from damage. Thus, S-312-d also
possesses an ability of protecting cerebroneuronal cells of those
in danger of being exposed to cerebral ischemia by, for example,
placed under a hypoxic and/or undernutritional conditions, as well
as treating or preventing dementia.
Thus, the present invention provides a pharmaceutical formulations
for the prevention and/or treatment of dementia, which comprises,
as an active ingredient, a therapeutically effective amount of
S-312-d.
Martin, J. B. and Gusella, J. F. (N. Engl. J. Med., 315: 1267-1276
(1986)) suggested that baclofen can decrease the excitation level
of neuronal cells excited by glutamate through the activation of
suppressive neuronal cells, whose transmitter is Y-amino butyric
acids, thereby preventing progress of Huntington's chorea disease
effectively. Baclofen, however, proved to inhibit the normal
glutamate release but not the ischemic release.
On the contrary, S-312-d is exclusively effective on ischemic
glutamate release, and therefor can be used clinically in the
prevention and/or treatment of disorders or symptoms caused by
massive release of cerebral glutamate. Examples of diseases which
can be treated with S-312-d include convulsion, epilepsy, diseases
caused by massive release of cerebral glutamate such as vascular
dementia, Altzheimer's disease, Huntington's chorea and senile
dementia, and necrosis of neuronal cells due to various causes
other than the cerebral glutamate.
As mentioned in the above, S-312-d inhibits sound-induced
convulsion in DBA/2 mouse. Although the mechanism by which the
convulsion is induced in DBA/2 mouse is not clear, it may be caused
by the activation of NMDA receptors with released glutamate (De
Sarro, G. B. et al., Br. J. Pharmacol. 93: 247-256 (1988)).
Croucher, M. J. et al.( Neuropharmacology, 23: 467-472 (1983))
reported that because NMDA receptor antagonist inhibited said
convulsion, the similar mechanism as the above can be also
responsible for the reflection-induced epilepsy in DBA/2 mouse.
The above-mentioned facts, that is, S-312-2 can inhibit the both of
a) the sound-induced convulsion, and b) the massive release of
cerebral glutamate, indicate that S-312-d is also useful as an
antiepileptic.
Thus, the present invention also provides an antiepileptic
formulation which comprises, as an active ingredient, a
therapeutically effective amount of S-312-d.
As previously described, S-312-d is a known compound which can be
prepared using any of known methods, for example, through a
resolution of a racemic S-312 which can be obtained according to
the procedures described in U.S. Pat. No. 4,703,051. However, it is
preferably prepared using the method disclosed in the Japanese
Patent Publication (KOKAI No.52890/1992) using, as a starting
material, 2-t-butyl 5-methyl
4,7-dihydro-3-isobutyl-6-methyl-4-(3-nitrophenyl)thieno[2,3-b]pyridine-2,5
-dicarboxylate which is disclosed in Japanese Patent Publication
(KOKAI) No.10087/1987. The above-mentioned references are herein
incorporated by reference.
S-312-d can be orally or parenterally administered to human or
animals and can be formulated into various forms in compliance with
the usage, including tablets, capsules, pills, granules, fine
granules, aqueous solutions, emulsions, vaginal suppository,
suppository, or the like. Formulations of the invention can be
prepared using any of the known methods in the art employing
conventional carriers or excipients therefor such as lactose,
sucrose, starch, cellulose, talc, stearic acid, magnesium stearate,
magnesium oxide, calcium sulfate, powdered gum arabic, gelatin,
sodium alginate, sodium benzoate, and the like. Solutions,
suspensions or injectable solutions are also employable by
dissolving S-312-d together with a solubilizing agent, into
distilled water, saline, Ringer's solution, or the like, or
suspending it in, for example, sesame oil.
The dose of S-312-d may vary depending on various factors such as
purpose and/or method of treatment, administration route,
conditions of the patient to be treated, and the like. However, it
may be administered to an adult at a dose of about 0.1-100 mg/day,
preferably about 0.5-50 mg/day, more preferably about 1-10
mg/day.
DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the change of regional (at hippocampal CIA) cerebral
blood flow in SHRSP before, during, and after ischemia caused by
BCAO. The relative changes in blood flow is plotted on the
ordinate, and the time (min) on the abscissa.
FIG. 2 shows the effect of Nimodipine (closed circle), Nilvadipine
(closed triangle), S-312-d (open triangle), and absence of
Ca-blocker (closed triangle), on the massive release of cerebral
glutamate during ischemia. In the figure, % changes of the
glutamate release to the resting release is plotted on the
ordinate, and the time (min) on the axis.
FIG. 3 shows the effect of S-312-d( closed circle), S-312-1 (open
circle), Nicardipine (open square), Nimodipine (closed triangle),
Flunarizine (closed square) against the ultrasonically-induced
lethal convulsion in DBA/2 mouse. The inhibitory rate (%) is
plotted on the ordinate, and the dose (mg/kg) on the abscissa.
PREPARATION 1
Methyl(+)-(4S)-4,7-dihydro-3-isobutyl-6-methyl-4-(
3-nitrophenyl)thieno[2,3-b1pyridine-5-carboxylate (S-312-d)
1.
4,7-Dihydro-3-isobutyl-5-methoxycarbonyl-6-methyl-4-(3-nitrophenyl)thieno[
2,3-b]pyridine-2-carboxylic acid
To a mixture of 108.8 g (0.224 mol) of 2-t-butyl 5-methyl
4,7-dihydro-3-isobutyl-6-methyl-4-(3-nitrophenyl)
thieno[2,3-b]pyridine-2,5-dicarboxylate, 67.0 g (0.447 mol) of
sodium iodide in acetone 500 ml was added dropwise 48.6 g (0.447
mol) of trimethylsilyl chloride at room temperature under stirring
and the mixture stirred for another 5 hr at room temperature.
To the mixture was then added dropwise 1,110 ml of ice-cold water,
and 50 ml of 10% Na.sub.2 S.sub.2 O.sub.3 solution, successively.
The crystalline precipitates are separated by filtration, washed
with water, and dried to yield 98.4 g (yield 98.2%; purity on the
basis of HPLC, about 96.1%) of the titled 2-carboxylic acid as
yellowish crystals, mp. 173 .degree.-174.degree. C.
IR (Nujol) max: 3306(NH), 2596(COOH), 1649(CO), 1342(NO.sub.2)
cm.sup.-1
.sup.1 H-NMR (d.sub.6 DMSO) .delta.:0.75(3H,d,j=7Hz),
0.85(3H,d,j=7Hz), 1.69(1H,m), 2.13(1H,dd,j=7.11Hz), 2.30(3H,s),
2.88(1H,dd,j=7,13Hz), 3.57(3H,s), 5.21(1H,s), 7.53-8.08(4H,m)
Elementary Analysis for C.sub.21 H.sub.22 N.sub.2 O.sub.6 S:
Theory: C, 58.59; H, 5.15; N, 6.51 Found : C, 58.29; H, 5.16; N,
6.40 .degree. . HPLC: t.sub.R 4.8 min (column: TSK-GEL ODS-120T, 5
.mu.m (4.0.times.250 mm); mobile phase: methanol, 0.5 ml/min;
detection: UV254 nm; 35 atmospheric pressure.
2. Methyl (+)-(4S)-4,7-dihydro-3-isobutyl-6-methyl
-4-(3-nitrophenyl)thieno[2,8-b]pyridine-5-carboxylate (S-312-d)
2.1. Cinchonine salt of S-312-d
A mixture of 97.1 g (0.224 mol) of 2-carboxylic acid obtained in
the above 1 and 65.9 g (0.224 mol) of cinchonine in 500 ml of
ethanol was heated under reflux for 10 min to dissolve the reaction
materials and the solution allowed to stand for 20 min at room
temperature. The crystalline precipitates are filtered, washed with
ethanol, and dried to yield 69.2 g (yield=42.8%) of S-312-d in the
form of cinchonine-addition salt as yellowish crystals, mp.
224.degree.-226.degree. C. (decomp.).
[.alpha.].sup.23.sub.D : +394.4.degree..+-.8.5.degree.
(C=0.513,DMSO)
IR (Nujol) max: 3226(NH), 1668(CO), 1342(NO.sub.2) cm.sup.-1.
2.2. S-312-d
To a solution of 64.4 g (0.089 mol) of cinchonine salt obtained in
the above 2.1. in 320 ml of methanol was added dropwise 14 ml (0.27
mol) of sulfuric acid with stirring. The temperature of the
reaction mixture was then elevated gradually and the mixture
refluxed for 15 min. To the cooled mixture was then added 650 ml of
ice-cold water and the crystalline precipitates were filtered,
washed with water, and dried to yield 47.5 g (yield=93%) of S-312-d
as yellowish crystals in the form of flat plates, mp
=173.degree.-175.degree. C.
[.alpha.].sup.23.sub.D : +274.3.degree..+-.3.0.degree. (C=1.019,
EtOH).
Elementary Analysis for C.sub.20 H.sub.22 N.sub.2 O.sub.4 S:
Theory: C, 62.16; H, 5.74; N, 7.25 Found : C, 62.04; H, 5.67; N,
7.28 .
IR(Nujol) max: 3305(NH), 1630(C=0), 1342 (NO.sub.2) cm.sup.-1
.sup.1 H-NMR (CDCl.sub.3).delta.: 0.72(3H,d,j=6Hz),
0.88(3H,d,j=6Hz), 1.60(1H,m), 2.02(1H,dd,j=7,15Hz),
2.13(1H,dd,j=7,15Hz), 2.37(3H,s), 3.63(3H,s), 5.25(1H,s),
6.22(1H,s), 7.35-8.08(4H,m).
HPLC: t.sub.R 15.5 min (column: SUMIPAX OA2000, 5 .mu.m
(4.0.times.250 mm); mobile phase: hexane-isopropanol (9:1), 1.0
ml/min; detection: UV248 nm; 40 atmospheric pressure.
The following experiments were conducted to evaluate the biological
activity of S-312-d.
Experiment 1
Effect of S-312-d on the Ischemic Release of Glutamate in
Hippocampal CAI Region
Test compound: S-312-d
Reference compound: nilvadipine, and nimodipine Animal: Five male
SHRSP bred at the Aburahi Laboratories (Shionogi) at ten to twelve
weeks of age were used in a group. Animals were fed CA-1 (Nihon
Clea) and tap water ad libitum.
A. Explanation of Occlusion of both Common Carotid Arteries
(BCAO)
A.1. Method
Hippocampus CAI region was made ischemia by the occlusion of both
common carotid arteries (BCAO) into rats as follows:
Rats were anesthetized with halothane (3%) and the cervical region
proximal to abdomen was incised to expose the common carotid
arteries. Both arteries were occluded with nylon threads with each
end exposed at the back of the neck through either of two holes of
a tube (double lumen PE tube, Natsume Seisakusho, & Co.).
Occlusion was induced in the both common carotid arteries by
pulling strongly each exposed end of threads. BCAO was stopped by
gradually pulling down the each end of threads passed under
arteries proximal to abdomen. During the ischemic period, the
halothane inhalation was stopped.
A.2. Determination of Regional Cerebral Blood Flow
The determination of the regional cerebral blood flow was conducted
using a laser doppler tissue flow meter (LFA-2, Biomedical Science,
Japan) with a needle-type sensor (450 .mu.m in diameter) inserted
into the hippocampal CA1 region opposite to the site where a
microdialysis probe had been fixed. The blood flow during BCAO was
estimated as almost negligible on the assumption that the blood
flow after the death of rat is "0".
B. Effect of S-312-d on the Ischemic Release of Cerebral
Glutamate
B.1. Administration of Test Compounds
S-312-d (0.3 mg/kg), nilvadipine (3 mg/kg), or nimodipine (3 mg/kg)
was dissolved into polyethylene glycol 400 and administered into
duodenum in each rat at 40 min before BCAO.
B.2. Measurement of Glutamate Release by Microdialysis
Under the anesthesia with halothane (1-2%), rats were fixed on
cerebral stereotaxic frame and equipped with an artificial
respirator (1 cc/100 gbw.times.60 times/min), while being
maintained at 37 .degree. C by settling a heating pad beneath the
lower abdominal region. A hole was drilled on the exposed skull and
a microdialysis probe (CMA/10, membrane length 2 mm, BAS, Sweden)
was inserted gently. Measurement was started after 3 hr from the
surgery in order to avoid the influence of tissue damage. The probe
was perfused with Ringer's solution at a flow rate of 2 .mu.l/min
by a microdialysis pump (CMA/100, BAS, Sweden). The dialysates were
collected for each 5-min perfusion and pooled. Sampling was started
at 10 min before the BCAO. BCAO was continued for 20 min when it
was stopped by loosening (occluding) threads to allow cerebral
circulation. The sampling was continued for 20 min after the
discontinuation of the BCAO. Samples were then subjected to
quantitative analysis for glutamate without further treatments.
B.3. Determination of Glutamate Concentration
The concentration of glutamate was determined by HPLC substantial
in accordance with the HPLC-ECD method of Donzanti et al. (Life
Sci. 43: 913 (1988)) The glutamate-containing sample was reacted
with o-phthlaldehyde/.beta.-mercaptoethanol solution for 2 min and
injected into a reverse-phase column and eluted under the following
conditions. The eluate was then analyzed for the presence of
glutamate by an electrochemical measurement of oxidation-reduction
current.
Conditions for the Measurements
Column: C 18, particle size: 3 .mu.m, 8 cm.times.4.6 mm
Temperature: 39.degree. C.
Mobile phase: a mixture of 0.1M phosphate buffer (pH 6.4), 0.13 mM
Na.sub.2 EDTA, and 22% MeOH
Flow rate: 1 ml/min
Results
Results are shown in FIGS. 1 and 2.
FIG. 1 shows the regional blood flow before, during and after the
ischemic period caused by BCAO. In the figure, the relative changes
in regional cerebral blood flow are plotted on the ordinate, and
the time (min) on the axis. As can be seen from the FIG. 1, the
regional blood flow decreased to an undetectable level during
ischemic period and is recovered to the pre-ischemic level after
the reperfusion.
FIG. 2 shows the influence of S-312-d on the massive release of
cerebral glutamate during BCAO. In the figure, the % change of the
release of glutamate to its basal release is plotted on the
ordinate, and the time (min) on the abscissa. As is clear from the
FIG. 1, S-312-d inhibited the massive release of glutamate during
BCAO without affecting the normal resting release of glutamate. On
the contrary, neither nilvadipine nor nimodipine shows significant
effect on the glutamate release during BCAO.
Thus, S-312-d inhibits specifically the massive release of
glutamate during cerebral ischemia but not the normal cerebral
release of glutamate in contrast to baclofen which inhibits the
normal release but not the ischemic release. From this evidence,
one can expect that S-312-d is clinically useful and can be applied
widely to the massive release of cerebral glutamate which results
from many causes, in addition to ischemia, such as over-excitation
of neurons during convulsion.
Experiment 2
Effects of S-312-d on the Ultrasonically-induced Convulsion in
DBA/2 Mouse
Test compound: S-312-d
Reference compound: methyl
(-)-(4R)-4,7-dihydro-3-isobutyl-6-methyl-4-(3-nitrophenyl)thieno[2,3-b]pyr
idine-5-carboxylate (hereinafter, referred to as S-312-1, an
optical isomer of S-312-d), Nicardipine, Nimodipine, and
Flunarizine.
Animal: Ten mice consisting of five each of male and female DBA/2
mice (weighing from 7 to 11 g) bred at the Aburahi Laboratories
(Shionogi) at three weeks of age were used in a group.
Method
One hour before the ultrasonic treatment, each mouse of test group
orally received S-312-d as a solution in polyethylene glycol 400.
To each mouse of control groups was orally administered either of
Nicardipine, as a solution in polyethylene glycol 400, S-312-1,
Nimodipine or Flunarizine, as a suspension in gum arabic.
Convulsion was induced in mouse by an ultrasonic treatment (70-90
db, 1 min) and the number of tetanic death was observed. ED50 value
of anti-convulsant effect of each compound was calculated by probit
method using the death from trauma as an indication.
Results
Results are shown in FIG. 3 and in Table 1 below. As is clear from
the FIG. 1 and Table 1, S-312-d and Flunarizine show the
dose-dependent anti-convulsant effect, while remaining typical
dihydropyridine Ca-blockers, i.e., Nicardipine, Nimodipine and
S-312-1, show anticonvulsive effect of less than 40%. It is notable
that the maximum effects of Nicardipine and Nimodipine are 30% and
40%, respectively, at a dosage ranging from 20-80 mg/kg, and that
of S-312-1 is 30% at a dosage ranging from 10-80 mg/kg.
TABLE 1 ______________________________________ Anticonvulsive
Effect in DBA/2 Mouse Group Compound ED50 (mg/kg)
______________________________________ Treated S-312-d 17.6 Control
S-312-1 30% at 10-80 mg/kg Nicardipine 30% at 20-80 mg/kg
Nimodipine 40% at 20-80 mg/kg Flunarizine 48.1
______________________________________
Experiment 3
Effects of S-312-d on the Delayed Neuronal Death During Cerebral
Ischemia
Test compound: S-312-d
Animal: eight to twelve (n=8-12) male SHRSP bred at the Aburahi
Laboratories (Shionogi) were used in a group. Animals were fed CA-1
(Nihon Clea) and tap water ad libitum.
Method
S-312-d was dissolved into polyethylene glycol 400 solution and
intraperitoneally administered to a SHRSP at 60 min before the
introduction of a global cerebral ischemia. Cerebral ischemia was
then caused in each rat for 20 min and stopped to allow cerebral
circulation in the same manner as the above Experiment 1. For the
histological observation on the delayed neuronal death, a fixation
of tissue was conducted by perfusing the animal from the left
ventricle using 4% paraformaldehyde at a flow rate of 10 ml/min by
means of perista pump (AC-2110, ATTA) on the 7th day from the
ischemia. After the removal of brain, cerebral region containing
hippocampus was embedded in paraffin, from which sliced
preparations of 4 .mu.m thick were prepared. Each cerebral
preparation was stained with hematoxylin-eosin and the density of
the living pyramidal neurons (n/mm) was determined by counting the
number of neurons per 1 mm length of the hippocampal CAI region
under a light microscope. The data were expressed as the mean
.+-.S.E.M. For the purpose of comparison, SHRSPs with or without
ischemic maneuvering were both treated with propylene glycol, in
exactly the same manner as the above. Results are shown in Table 2
below.
TABLE 2 ______________________________________ Density of Neurons
(n/mm) Group <40 40-80 80-120 >120 mean .+-. S.E.M
______________________________________ Normal 0 0 1 7 160 .+-. 9
Control 2 4 6 0 69 .+-. 10* Treated (I) 3 4 2 3 73 .+-. 12** (II) 2
1 4 6 114 .+-. 17*** ______________________________________ Normal
group (n = 8), control group (n = 12), treated group (I) (n = 12,
S312-d 0.01 mg/kg) and treated group (II) (n = 13, S312-d at the
dosage o 0.1 mg/kg) *p < 0.05, **p < 0.01; significantly
different from normal group. ***p < 0.01; significantly
different from control group.
As can be seen from the above Table 2, there is a marked neuronal
necrosis in the control group compared with the normal group. It is
also clear from the table that the pretreatment with S-312-d is
significantly effective for the protection of cerebral neurons
against ischemic necrosis of cells.
Experiment 4
Single Dose Toxicity
A 5% Gum Arabic suspension of S-312-d was orally administered to
6-week-old Jcl:SD male rats (n =6) at a S-312-d dosage of 5000
mg/kg each. During a 14 day observation, they were all alive.
The following formulation examples are illustrative only and are
not intended to limit the scope of the invention.
EXAMPLE 1
Powders are prepared using the following ingredients:
______________________________________ Quantity (mg)
______________________________________ S-312-d 4 Corn starch 93
St--Mg* 1 Talc 1 Kp80** 1 Total 100 mg
______________________________________ *St--Mg = magnesium stearate
**Kp80 = Carplex 80# (Shionogi Seiyaku Kabusiki Kaisha, Japan)
The above ingredients are mixed thoroughly.
EXAMPLE 2
Hard gelatin capsules are prepared using the following
ingredients:
______________________________________ Quantity (mg)
______________________________________ S-312-d 4 Corn starch 93
St--Mg 1 Talc 1 Kp80 1 Total 100 mg
______________________________________
The above ingredients are mixed thoroughly filled into hard gelatin
capsules in 100 mg quantities.
EXAMPLE 3
Tablets are prepared using the following ingredients:
______________________________________ Quantity (mg/tablet)
______________________________________ S-312-d 4 Lactose 56 Corn
starch 28 HPC* 1.5 CMC--Ca** 10 St--Mg 0.5 Total 100 mg
______________________________________ *HPC = hydroxypropyl
cellulose **CMC--Ca = calcium carboxymethyl cellulose
The above ingredients are mixed thoroughly and the resultant
powders are compressed on tablet machine to yield tablets each
weighing 100 mg.
* * * * *